Single gene mutations can be used specifically to alter any given macromolecule or regulatory process involved in the function of excitable cells. A proper collection of mutants should enable us to dissect these structures and events. This proposed research involves Drosophila mutants defective in membrane excitability and in synaptic transmission. There are a number of behavioral mutants that are known to have altered nerve excitabilities. Among them, nap(ts) and para(ts) block action potentials. Although these mutants have been fairly well characterized genetically, physiological analysis of their membrane properties can be further advanced. Physiological characterization will be conducted on neurons both in situ and in culture with intracellular microelectrode techniques. Fusion of dissociated neurons has been carried out and will be further advanced so that intracellular recordings as well as other electrophysiological techniques can be more readily applied to large fused cells. Voltage clamp analysis will be performed on the larval muscle to obrain a rigorous description of the membrane conductances. Since Drosophila muscles lack voltage-dependent Na conductance, we will concentrate on the K conductances in the wild type and in Sh, which presumably have altered K conductance in nerve. We will use both single-gene mutations and genetic interactions (either intragenic or intergenic) to help analysis of current models of ionic channels. The mutant shi(ts) has been shown to block synaptic transmission. Experiments are designed to determine the primary defect responsible for the transmission block. Aspect of transmitter release, storage and synthesis will be investigated by using combination of electrophysiological, ultrastructural and biochemical techniques. Analysis of shi(ts) may reveal interdependence between synaptic events such as Ca++ influx, vesicle formation and other membrane-bound processes common to many cell types.